We postulate that there is extensive crosstalk between hematopoietic signal transduction pathways. We propose that this crosstalk helps to regulate the activation and termination of signals emanating from the surface of activated hematopoietic cells. To begin to address this hypothesis, two constitutively active receptors (cEpoR and CD8-cEyk), known to activate the JAK/STAT pathway, were introduced into murine hematopoietic (Ba/F3) cells. Both caused factor independence and transformation, as a result of activation of the JAK/STAT and Ras/Raf pathways. However, signaling events initiated by cEpoR and CD8-cEyk were clearly different. To further evaluate these differences, we have developed an in vivo assay in Drosophila blood cells. We take advantage of the fact that signaling pathways are remarkably conserved among metazoans. cDNAs for both receptors have been placed under the control of Gal4 transcriptional regulatory elements, and injected into Drosophila embryos. Transgenic flies were isolated and crossed with Drosophila lines expressing Gal4 protein under the control of a heat shock promoter. Upon heat shock, expression of the vertebrate receptors is induced, resulting in leukemia-like proliferation of blood cells and formation of melanotic tumors. This offers a powerful, novel assay to probe signal transduction circuits in vivo. We will further refine this assay, and determine the participation of the JAK/STAT, Ras/Raf, NF-kappaB and JNK pathways in hemocyte proliferation and tumorigenesis in genetic experiments. We will test whether loss of function mutants in these pathways can suppress the melanotic tumor phenotype. In addition, we will use this system to characterize a hyperphosphorylated STAT5 mutant that we have recently generated. We have shown that this STAT5 mutant is resistant to dephosphorylation. We expect to find that it confers a gain of function phenotype. Finally, we will exploit a genetic screen in Drosophila to isolate novel genes that interact with known signal transduction pathways to suppress the melanotic tumor phenotype. Viable and fertile flies, which all show melanotic tumors, will be crossed with mutant stocks carrying chromosomal aberrations, P-element insertions, or chemically- induced point mutations. Suppression of the melanotic tumor phenotype in the F1 generation will serve as the criterion to identify accessory factors that modulate known hematopoietic signal transduction pathways. This series of experiments should produce new insights into hematopoietic signal transduction that could not be obtained from standard biochemical experiments, or from gene manipulations in mice.